Determination of Osteoblast Cell Viability and Histological Changes of Samples Obtained from Different Implant Drills during Osteotomy

Statement of the Problem: The bone particles collected during osteotomy could be used as autogenous bone graft materials for dental implant surgery. Different factors such as drill design may influence its clinical viability. Purpose: This study examined the effect of drill design on the osteoblast viability and histopathology parameters of bone collected during the preparation of dental implant site. Materials and Method: In this experimental study, 90 samples were obtained from three different bone drilling systems including Bego, Implantium, and Dio during fixture installation in patients requiring treatment at the Department of Periodontology, Dentistry University Hamedan. The MTT (3-4,5-Dimethylthiazol2,5-diphenyltetrazolium bromide) was used to determine percentage of cell viability. Samples were fixed in 10% formaldehyde for histological evaluation. Then, they were kept in 10% EDTA solution for 4 weeks for decalcification. The provided slides were evaluated regarding bone structure and osteocytes counts for assessment of viability. Tukey test and SPPS 21 software were used for statistical analysis. Results: The result showed the viability of osteoblast obtained by Dio (0.45±0.04) was significantly better than Bego (0.37±0.05) and Implantium (0.37±0.04) systems. In histopathological evaluation, the grafting material obtained by Dio presented the best osteoblast morphology. Conclusion: It might be concluded that drill geometry has significantly influenced the viability of bone particles collected during the preparation of implant sites .Moreover, characteristic geometry alone cannot represent the performance of a particular drill, and several geometric features should be concerned. The results of this study showed that the geometry of the Dio drill was the best considering the viability and histopathological evaluations.


Introduction
There is an increasing demand for dental implant placement considering patients satisfaction in recent years. This increase might be related to particular reasons rather than replacing lost teeth; including esthetic aspects, speech ability, chewing, self-confidence, and better life quality. On other hand, dentists are interested in dental implants since there is no need to prepare tooth, and increased retention, and stability especially in fully edentulous cases are among the advantages of this treatment modality [1][2]. The success of implant treatment is related to adequate available bone at the implant site and this issue would influence the long-term prognosis. In addition, there are some techniques to increase the width and height of alveolar ridge including distraction osteogenesis, grafting, and bone splitting, and guided bone regeneration (GBR). Currently, autogenous bone graft is known as the gold standard for GBR [3][4].
The type of surgical drill and drilling method are the most important items in dental implant treatment. Eriksson and Albrektson [5] reported that bone becomes more damaged during drilling especially concerning thermal damage. They reported that the threshold level of bone vitality is 47°C during drilling and less than one minute. Decreased cutting efficacy and frictional heat are the consequences of using cutting instruments frequently [5]. The heat produced during drilling is related to pressure, shape, and the size of drill as well as drilling time. The factors related to heat production are reported as drilling speed, acquired depth, geometry of drill, and sharpness of drill [6].
In recent years, the use of stainless steel drills has been successful in dentistry [7]. Ceramic drills have some advantages like resistance against high temperature, corrosion, abrasion, and lower tendency for reaction with chemical materials. Despite all these, the application of this kind of drills is limited because of their lower resistance against mechanical shocks, potential fracture tendency, and low thermal conductivity [7][8]. It is reported that the efficacy of a drill is related to design and mechanical properties. Employment of different drill types would result in different properties of collected bone [9]. The osteoblasts create the protein matrix of bone structure. These cells usually construct bone while the osteoclasts are responsible for constant bone remodeling [10].
An in vitro study showed that bone milling resulted in decreased osteoblast cells [11]. Therefore, basic conditions for cell viability are drill selection, proper operation, and control bacterial infection. The utilization of different drills alters the osteoprotegerin [12]. Osteoprotegerin is a protein secreted from osteoblasts. Different drills usage also causes changes in receptor activator of nuclear factor kappa-Β ligand (RANKL) protein, which plays an important role in bone dynamic. RANKL is important for osteoclastic activity and bone remodeling cycle. Therefore, the type of drill is considered as an imperative factor [12].
Concerning the limited evidence on this topic, this study was conducted to assess the osteoblasts viability when different drill designs are used during osteotomy.

Materials and Method
In this experimental study, 90 samples were collected using three different drilling systems (30 samples for each drill type) from patients with edentulous ridge who were referred for tooth implant surgery.
The inclusion criteria were defined as negative history of any periodontal disease and presence of good oral hygiene. The exclusion criteria were delineated as pregnancy, smoking or alcohol consumption, uncontrolled systemic disease, unacceptable oral hygiene, his-tory of radiotherapy or chemotherapy, taking immunosuppressive medications, and long-term steroid therapy.
The Misch classification was used to define the bone types. Correspondingly, D2 was considered as dense cortical bone with large coarse trabecular bone, and D3 was considered as thin cortical bone and dedicated trabecular bone pattern. The density of all collected samples was the same regarding the tactile sensation [13].
As illustrated in Figure 1, Dio (Dio co., DAEGU, South Korean), Implantium (Dentium co., Chongju, Korea), and Bego (Bego co., Bremen, Germany) drills were employed in this study. Drilling speed was 800 rpm and equal for all groups. Patients used 0.1% chlorhexidine rinse for 2 minutes before surgery in order to reduce bacterial contamination. The collected bone samples were kept in Normal Saline solution (0.90% w/v) and then sent to laboratory on ice [14].
The MTT (3-4,5-Dimethylthiazol2,5-diphenyltetrazolium bromide) was used to determine the percentage of cell viability. MTT is a sensitive method for evaluating osteoblasts proliferation and cell viability through oxidation of MTT by mitochondrial dehydrogenase. The Cell Titer 96 (promega, madison, WI, USA) (MTC) was used to determine the cell viability [15].
The samples were fixed in 10% formaldehyde for histological evaluation. Then, they were kept in 10% EDTA for 4 weeks for decalcification; the EDTA solution was being changed weekly. The provided slides were evaluated for bone structure and osteocytes count, which indicate the viability.
Tissue processing was done manually after sample fixation with 10% formalin. The samples were softened and decalcified with 10% nitric acid for one hour. All samples were then rinsed to exclude the acid.
The samples were kept at five different ethylic alcohol solutions (50%, 70%, 80%, 96%, and 100%) respectively, in order to dehydrate the excess water of fixation stage. Then, for clearing, all samples were kept in Xylenol for one to two hours and this was repeated with the new Xylenol. In impregnation stage, the samples were settled in melted paraffin at 56°C for 24 hours.
The paraffin was changed to achieve better impregnation. Then, the samples were transferred at embedding stage. The utilized mold was made of aluminum. Rotary micro tom (SLEE) was used for sectioning at 5μm width serial section. Sections were floated at 48-50°C water in a dark barrel to distinguish wrinkle tissue better. Sections were located at slide impregnated with albumin adhesive. Then, slides were located on the hot plate to dry. Hematoxylin -Eosin was used for staining. Slides were evaluated with light microscope after mounting.
Tukey test and SPPS 21 software were used for statistical analysis.

Results
A total of 90 patients, 53 male and 37 female individuals, were enrolled in this study and divided into 3 groups. All the operations were performed by a single periodontist in posterior regions. All collected samples with different drills were examined for histopathological and viability assessment.

Cells viability evaluation
The viability was statistically significant in Dio group compared to other groups (p value 0.001) ( Table 1).
As the result of Tukey test, there was a significant difference among Dio drill samples and the others in favor of viability (p value 0.001). However, there were not any significant differences between Bego and Implantium drill samples (p value 0.948 for differences between Bego and Implantium). As illustrated in   (Table 3).

Discussion
Autogenous bone graft is the best graft material that has some advantages such as osteogenicity, osteoconductiv-    of which is osteocytes in calcified matrix [17].
In an animal study, researchers concluded that the collected bone presented more osteoactivity and absorption characteristics [18]. When these collected bony particles were grafted into dehiscence or fenestration implant sites, the results were successfully stable and well maintained [17,19].
According to our study, Dio drill samples showed Park et al. [23] reported that low drilling speed leads to a large particle size. Marzook et al. [24] suggested that high speed drilling of dense bone in addition to irrigation results in less temperature change and better cell viability. Moreover, bone viability is better maintained while using lower speed. In brief, the drill diameter is inversely related to temperature changes [23].
In a systematic review, low-speed drilling without irrigation was compared with conventional drilling. It was concluded that low-speed drilling without irrigation presented better results [25].
There are three reasons for significant better success rates of smaller particles compared to large ones; they include increased surface area, increased osteoclasts activity and osteogenic activity, and finally osteogenesis stimulation [23]. The different drill geometry designs, such as web, thinning, and flute, may influence the bone formation. In this study, we used straight tapered Bego drills, parallel twist Implantium drills, and twiststepped-tapered Dio drills. These implant system drills have detailed characteristics presented in Table 3.
The lesser web diameter results in better formation and discharge of bone chips; this is the same as the results of our study considering the better viability values and histopathological structure. Park et al. [23] reported that drilling geometry is one of the most important fact- Finally, Chen et al. [27] suggest that faster new bone formation of implant site is related to a reduced zone of osteocyte death that should be considered.
Despite the fact that piezosurgery is contributed with greater cell viability compared to traditional drills, some aspects such as speed of surgery may affect the technique selection for implant site osteotomy [28]. Chen et al. [29] introduced a novel osteotomy preparation technique to preserve implant site viability and enhance osteogenesis. Unique design of the cutting flutes with no irrigation and low-speed was used.
The results of the present study may also be used to design implant drills for better cell viability based on the used drills characteristics. Dio drills are twist, stepped, and tapered. We suggest assessing different drilling speeds by different implant systems for future studies. It is also better to use control drills in these studies.

Conclusion
It may be concluded that drill geometry significantly influenced the viability of bone particles collected during the preparation of implant sites. Moreover, characteristic geometry alone cannot represent the performance of a particular drill, and several geometric features should be concerned. The results of this study showed that the geometry of the Dio drill was the best considering the viability and histopathological evaluations.